The present invention relates to a method for the enzymatic synthesis of isotopically labeled aldose and ketose phosphates. More specifically, a method is provided for the synthesis of a number of aldose and ketose phosphates and their derivatives, labeled with .sup.13 C at any one of a number of single sites or synthetically-related sites, starting from .sup.13 C-enriched pyruvate and using enzymes of the glycolytic pathway.
Present methods for the synthesis of labeled carbohydrates utilize a method involving the serial condensation of .sup.13 C-cyanide with an aldose to produce an aldose one carbon longer than the original aldose which is labeled at the reducing carbon. However, this method suffers from a number of disadvantages and limitations which decrease its utility. For example, to produce an aldose two carbons longer than the original aldose involves a lengthy purification step, via ion exchange and absorption chromatography, and a second condensation with cyanide to produce the longer aldose. Further, this method may be utilized for the labeling of only one or both of the two carbons at the reducing end of the molecule, and provides an overall yield of only 50-60%.
Methods are available for the enzymatic inter-conversion and condensation of isotopically labeled dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate to yield labeled D-fructose-1,6-diphosphate. The enzyme involved in this condensation, muscle aldolase, is also tolerant of accepting a wide variety of other 1-4 carbon aldehydes as substrates, making feasible the specific isotopic enrichment of a variety of 4-6 carbon ketose phosphates from isotopically-enriched dihydroxyacetone phosphate. Purified enzyme preparations are also available commercially for the conversion of the product ketose phosphate to phosphate analogs of D-glucose, D-mannose, D-galactose, D-ribulose and D-ribose, the latter via the pentose phosphate shunt, and other aldoses and ketoses.
At the other end of the glycolytic pathway, .sup.2 H and .sup.13 C enriched phospho(enol)pyruvate has been prepared in relatively high yield from isotopically labeled pyruvate using the enzyme phospho(enol)pyruvate synthetase. However, because of the large positive free energy change from pyruvate to dihydroxyacetone phosphate, via the enzyme catalyzed reaction of the glycolytic pathway, no method has been devised for the isotopic enrichment of aldose and ketose phosphates from labeled pyruvate.